This invention relates to the field of kidney disease (i.e., kidney disorder) characterized by glomerulonephritis and/or fibrosis. In particular, this invention relates to the use of xcex11xcex21 integrin receptor inhibitors in kidney disorders. Further, this invention relates to the use of xcex11xcex21 integrin inhibitors in combination with TGF-xcex21 inhibitors in kidney disorders.
In the United States, approximately 12,000 people currently live with Alport syndrome. This inherited disorder results in progressive renal disease that is only treatable by dialysis and kidney transplant. Transplanted kidneys are usually rejected. Thus, alternative treatments are needed. However, there is currently no treatment that addresses the mechanism of the disease onset or progression. Thus, what is needed is a treatment method that attacks the mechanism of disease onset and/or progression, one that could substantially slow disease conditions, such as renal glomerulonephritis and renal fibrosis.
A number of kidney diseases are associated with alterations in matrix homeostasis, where the delicate balance of synthesis and turnover of structural molecules is interrupted. As one example, Alport syndrome is a disease resulting in progressive renal failure and is associated with sensorineural hearing loss. Male carriers are most affected and ultrastructural studies reveal abnormalities in the glomerular basement membrane (GBM) of affected individuals. About one in 20,000 people have Alport syndrome, making the disease one of the more prevalent known genetic disorders. See, for example, Atkin et al., xe2x80x9cAlport Syndromexe2x80x9d In R. W. Schrier and C. W. Gottschalk (Eds.), Diseases of the Kidney, 4th ed., Chap. 19, Little Brown, Boston, pp. 617-641, 1988. X-linked Alport syndrome is caused by any of a series of mutations in the collagen 4A5 gene (Barker et al., Science, 248:1224-1227, 1990). At least 60 different mutations in the gene have been identified. The autosomal form of Alport syndrome displays the same range of phenotypes as the X-linked form and results from mutations in either basement membrane collagen gene 4A3 (COL4A3) or 4A4 (COL4A4). See, for example, Lemmink et al., Hum. Mol. Gen., 3:1269-1273, 1994, and Mochizuki et al., Nature Genet., 8:77-81, 1994. Other diseases of the basement membrane include Goodpasture syndrome, which is due to an acute autoimmune response directed against an epitope on the NCl domain of collagen 4A3 (Hudson et al., Kidney Int., 43:135-139, 1993), and diffuse leiomyomatosis, a benign smooth muscle tumor that is associated with a deletion of both collagen 4A5 and 4A6 (Zhou et al., Science, 261:1167-1169, 1993).
Basement membranes are specialized extracellular structures associated with nearly every organ and tissue in the body. They are usually found at the boundary between cells and connective tissue, but may also be found between epithelial and endothelial cells, as is the case in a kidney glomerulus (i.e., cluster of capillaries). The predominant building blocks of basement membranes include type IV collagen, laminin, heparin sulfate proteoglycan, entactin, and sometimes fibronectin and type V collagen. The most highly represented component in all basement membranes is type IV collagen, a distinct collagen type found only in basement membranes. In its native form, type IV, like all collagens, is composed of three collagen molecules assembled in a triple helix consisting of distinct combinations of the six alpha chains (4A1-4A6). The 4A1 and 4A2 chains (also referred to as the xcex11(IV) and xcex12(IV) chains) are the most common (Timp1, Eur. J. Biochem., 180:487-502, 1989). Type IV collagens differ from interstitial collagens in a number of ways. The helical structure of the alpha chain association does not strictly adhere to the glycine-X-Y motif observed in other collagens; it contains 3-hydroxyproline rather than 4-hydroxyproline, and is rich in carbohydrate. The resulting superstructure of collagen is a chicken wire-like network of basement membrane collagen. This network is the foundation onto which the accessory molecules (laminin, heparin sulfate, etc.) bind.
Basement membranes are very heterogeneous structures, which accounts for their diverse functional properties. The complexity of these structures is still poorly understood. Several novel basement membrane collagen chains (alpha 3, 4, 5, and 6 chains) were only recently discovered. See, for example, Gunwar et al., J. Biol. Chem., 266:15318-15324, 1991; Hostikka et al., Proc. Natl. Acad. Sci. USA, 87:1606-1610, 1990; Butkowski et al., J. Biol. Chem., 262:7874-7877, 1987; and Zhou et al., Science, 261:1167-1169, 1993. Interestingly, the novel chains have been found only in certain tissues (e.g., the glomerulus of the kidney, the Decimet""s membrane of the eye, the lens, the skin, the lung, the testis, and the cochlea). See, for example, Kleppel et al., Am. J. Pathol., 134:813-825, 1989, and Tryggvason et al., Kidney Int., 43:38-44, 1993. The role of these novel chains in basement membrane assembly and function is currently unknown. It is believed that these novel basement membrane collagens form separate networks distinct from the networks of collagen types 4A1 (xcex11(IV)) and 4A2 (xcex12(IV)).
Kidney glomerular basement membranes (GBMs) are integral to the ultrafiltration process (i.e., in which blood is filtered to remove metabolites for excretion in the form of urine, for example). Alport syndrome results in a massive accumulation of extracellular matrix and a compromised basement membrane, resulting in focal and segmental glomerulonephritis (i.e., inflammation of the capillary loops in the glomeruli of the kidney), which ultimately results in fatal uremia (i.e., excess urea in the blood as a result of kidney failure). Many of the same extracellular matrix molecules (e.g., collagen type I, fibronectin, laminin, and collagen type IV) also progressively accumulate in the GBM of patients with IDDM (insulin dependent diabetes mellitus) nephritis. In this disease, however, the GBM thickens, but lacks the focal thinning and splitting (segmenting) of the GBM, which is characteristic of Alport syndrome.
The integrins are a family of heterodimeric transmembrane glycoprotein receptors that bind to components of basal lamina and extracellular matrix. They function as adhesion molecules involved in cell aggregation and in anchoring cells to basal lamina. They also transduce signals to the nucleus, and are involved in modulating gene expression, particularly gene expression for cell migration and cell differentiation (Hynes, Cell, 69:11-25, 1992). Over 20 different integrin receptors are known, which include about 14 different alpha subunits and about 8 different beta subunits (DeSimone, Curr. Opin. Cell Biol., 6:747-751, 1994).
In the renal (kidney) glomerulus, there are distinct sets of integrin receptors. These are associated with either the mesangial matrix (i.e., a membrane that helps support the capillary loops in a kidney glomerulus) or visceral epithelial cells (Patey et al., Cell Adhesion Commun., 2:159-167, 1994). The most prevalent integrin receptor on adult glomerular visceral epithelial cells is the xcex13xcex21 heterodimer (Adler, Am. J. Pathol., 141:571-578, 1992; and Patey et al., Cell Adhesion Commun., 2:159-167, 1994). The xcex25 subunit has been shown to be expressed in adult visceral epithelial cells (Yamada et al., Cell Adhesion Communic., 3:311-325, 1995), and the xcex11, xcex13, xcex15, xcex1V, xcex21, and xcex23 integrin receptors are expressed developmentally during kidney morphogenesis (Korhonen et al., Lab. Invest., 62:616-625, 1990; Wada et al., J. Cell. Biol., 132:1161-1176, 1996; and Yamada et al., Cell Adhesion Communic., 3:311-325, 1995). The xcex11xcex21 heterodimeric integrin receptor is the only integrin receptor identified on the surface of mesangial cells in the renal glomerulus.
A gene knockout mouse at the xcex13 integrin receptor subunit has been produced. The offspring die of kidney dysfunction shortly after birth (Kreidberg et al., Dev., 122:3537-3547, 1996). The ultrastructural pathology of the GBM in the neonates of this model is remarkably similar to that observed in advanced Alport syndrome. The basement membrane appears rarefied (i.e., irregularly thickened, thinned, and split) and the foot processes of the visceral epithelial cells appear fused. Since one ligand for the xcex13xcex21 receptor is type IV collagen (Krishnamurti et al., Lab. Invest., 74:650-657, 1996; and Rupprecht et al., Kidney Int., 49:1575-1582, 1996), and since this receptor localizes along the plane of contact between visceral epithelial cells and the GBM (Baraldi et al., Nephron, 66:295-301, 1994), the observations listed above for the xcex13 integrin knockout support a model where such integrin/ligand interactions play an important role in basement membrane development.
In a normal animal, the type IV collagen in embryonic glomerular basement membrane (GBM) up to the time of birth is comprised entirely of the xcex11(IV) and xcex12(IV) chains (referred to as the classical collagen chains). Shortly after birth, a developmental switch occurs where the xcex13(IV), xcex14(IV), and xcex15(IV) chains (referred to as the novel collagen chains) are clearly detectable in the GBM, and the xcex11(IV) and xcex12(IV) chains become predominantly localized to the mesangial matrix (Miner and Sanes, J. Cell. Biol., 127:879-891, 1994).
In the adult kidney a thin layer of GBM comprised of the xcex11(IV) and xcex12(IV) chains lies adjacent to the endothelial cell layer, while the majority of the full thickness of the GBM is comprised of the xcex13(IV), xcex14(IV), and xcex15(IV) chains (Desjardins and Bendayan, J. Cell. Biol., 113:689-700, 1991; and Kashtan et al., J. Clin. Invest., 78:1035-1044, 1986). There is biochemical evidence suggesting that the two different sets of collagen chains form separate networks (Kleppel et al., J. Biol. Chem., 267:4137-4142, 1992). In familial nephritis, null mutations (i.e., mutations that destroy gene expression) in either the xcex13(IV), the xcex14(IV), or the xcex15(IV) genes results in the absence of all three chains in the GBM, presumably due to obligatory associations in macromolecular assembly of the GBM suprastructure. This results in the presence of xcex11(IV) and xcex12(IV) chains throughout the full thickness of the GBM. Thus, type IV collagen receptors on the surface of visceral epithelial cells in the Alport kidney (i.e., the kidney of an individual with Alport syndrome) are in direct contact with a GBM of uncharacteristic type IV collagen chain composition. At least one study has addressed the relative ability of the visceral epithelial cells to adhere to type IV collagen with these different compositions, and found that they adhere significantly better to basement membrane comprised of the novel chains when compared directly with the classical chains xcex11(IV) and xcex12(IV). This adhesion could be blocked with antibodies against the xcex13 integrin receptor.
A mouse model for the autosomal form of Alport syndrome was created by targeted mutagenesis of the COL4A3 procollagen gene (Cosgrove et al., Genes Dev., 10:2981-2992, 1996). The animal model develops a progressive glomerulonephritis with the onset of proteinuria at about four weeks of age and a mean age of death from renal failure at about 8.5 weeks in the inbred 129 Sv/J background. Ultrastructural changes in the GBM are observed as early as one week of age, and throughout the GBM of most glomeruli by 3 weeks of age, long before the onset of proteinuria. Extracellular matrix components including laminin-1, heparin sulfate proteoglycan, fibronectin, and entactin accumulate in the GBM. This mouse is referred to herein as the xe2x80x9cAlportxe2x80x9d mouse.
The accumulation of extracellular matrix in the GBM and the mesangium as a function of renal disease progression is a feature shared by a variety of glomerular diseases, both in patients and in experimental animal systems. See, for example, Goyal and Wiggins, Am. Soc. Nephrol., 1:1334-1342, 1991; Wilson et al., Contrib. Nephrol. Basel, Karger, 118:126-134, 1996; Razzaque et al., Clin. Nephrol., 46:213-215, 1996; Yoshioka et al., Kidney Int., 35:1203-1211, 1989; and Klahr et al., N. Engl. J. Med., 318:1657-1666, 1988. In diabetes, the primary mediator of this effect is thought to be prolonged exposure to non-enzymatically glucosylated serum proteins resulting from chronic high glucose levels (Doi et al., Proc. Natl. Acad. Sci. USA, 89:2873-2877, 1992; and Roy et al., J. Clin. Invest., 93: 483-442, 1994).
For the majority of progressive glomerular disorders, over-production of the transforming growth factor TGF-xcex21 seems to be closely associated with the accumulation of extracellular matrix leading to fibrosis (i.e., the formation of fibrous tissue). See, for example, Border and Ruoslahti, Nature (London), 346:371-374, 1990; Yang et al., J. Am. Soc. Nephrol., 5:1610-1617, 1995; and Yamamoto et al., Kidney Int., 45:916-927, 1994. In an animal model for autoimmune nephritis, injection with either antibodies to TGF-xcex21, or antisense oligonucleotides to the corresponding mRNA inhibited the progressive glomerulonephritis and the accumulation of extracellular matrix (Border et al., Nature (London), 346:371-374, 1990; and Akagi et al., Kidney Int., 50:148-155, 1996).
The half-life of basement membrane collagen in the GBM of the rat has been estimated at between 16 and 40 days based on pulse-chase studies with 3H-proline (Daha et al., Nephron. 22:522-528, 1978). This is very slow in relation with the turnover of heparin sulfate proteoglycans (txc2xd=20 hours) or other sulfated macromolecules in the GBM (txc2xd=20-60 hours). The accumulation of basement membrane proteins in the GBM of the Alport mouse model (Cosgrove et al., Genes Dev., 10:2981-2992, 1996) is likely the net effect of changes in both the synthesis and the degradation of these proteins. Of the proteases involved in the turnover of both GBM and mesangial matrix, the most characterized are the metalloproteinases MMP-2 (72 kD collagenase) and MMP-9 (92 kD collagenase), as well as MMP-3 (stromolysin-1). These enzymes will degrade type IV collagen, in addition to a variety of other extracellular matrix components.
Mesangial cells (and probably other glomerular cell types) also produce natural inhibitors to the metalloproteinases. These are called TIMP""s (for Tissue Inhibitors of MetalloProteinases). These are relatively low molecular weight glycoproteins. Of these, TIMP-1 is specific for stromolysin-1 and MMP-9, while TIMP-2 and TIMP-3 will inhibit MMP-2 (Goldberg et al., Proc. Natl. Acad. Sci. USA, 86:8207-8211, 1989; Staskus et al., J. Biol. Chem., 266:449-454, 1991; and Stetler-Stevenson et al., J. Biol. Chem., 264:17374-17378, 1989).
Modulation of the metalloproteinases and their corresponding inhibitors likely play a role in maintaining appropriate levels of GBM turnover. While little is known regarding the regulation of the genes encoding these proteins in the glomerulus, signal transduction via integrin receptor/ECM (extracellular matrix) interaction may be a key aspect in this process.
There remains a need for animal models for Alport syndrome, particularly one in which the disease progression is slowed significantly. There also remains a need for new therapies to treat kidney diseases associated with mesangial matrix expansion, and progressive matrix accumulation in the glomerular basement membrane and the tubulointerstitium, including Alport syndrome and insulin dependent diabetes mellitus, for example.
The present invention provides various treatment methods for treating or limiting (i.e., delaying the onset of, slowing the progression of, and/or reversing) a kidney disorder in a patient (preferably, a mammal, and more preferably, a human). The kidney disorder preferably includes renal glomerulonephritis, renal fibrosis, or both. These conditions can be associated with, for example, Alport syndrome, IDDM nephritis, mesangial proliferative glomerulonephritis, membrano proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, and renal insterstitial fibrosis.
In one embodiment, the method involves administering to the patient an effective amount of an xcex11xcex21 integrin receptor inhibitor. This xcex11xcex21 integrin receptor inhibitor can be a blocking agent that binds to the xcex11xcex21 integrin receptor binding site on the surface of a kidney cell. The blocking agent can be an at least 9-mer peptide fragment of a protein selected from the group consisting of laminin, fibronectin, entactin, and collagen type 4. Alternatively, the blocking agent can be an antibody. Other agents that inhibit (i.e., inactivate) the xcex11xcex21 integrin receptor by other mechanisms can also be used.
In another embodiment, the method involves administering to the patient an effective amount of a TGF-xcex21 hibitor in addition to the xcex11xcex21 integrin receptor inhibitor. These inhibitors can be administered simultaneously (e.g., as in a mixture) or sequentially. The TGF-xcex21 inhibitor can be an agent that irreversibly binds to TGF-xcex21 and inhibits its ability to bind with its receptor. Alternatively, the TGF-xcex21 inhibitor can be an agent that inhibits the ability of TGF-xcex21 to transduce signals to the nucleus of a kidney cell. The latter type of inhibitor is preferably a calcineurin inhibitor, such as tacrolimus (commercially available as FK506). Other agents that inhibit (i.e., inactivate) TGF-xcex21 by other mechanisms can also be used.
Preferably, the present invention provides methods for delaying the onset of and/or slowing the progression of Alport syndrome in a patient. In one embodiment, this method involves administering an effective amount of an agent that inhibits signal transduction through an xcex11xcex21 integrin receptor of a kidney cell. In another embodiment, this method involves blocking an xcex11xcex21 integrin receptor binding site on the surface of a kidney cell of the patient. These methods can be further enhanced by administering to the patient an effective amount of a TGF-xcex21 inhibitor.
Preferably, the present invention also provides methods for delaying the onset of and/or slowing the progression of kidney disease in insulin dependent diabetes mellitus in a patient. In one embodiment, this method involves administering an effective amount of an agent that inhibits signal transduction through an xcex11xcex21 integrin receptor of a kidney cell. In another embodiment, this method involves blocking an xcex11xcex21 integrin receptor binding site on the surface of a kidney cell. These methods can be further enhanced by administering to the patient a TGF-xcex21 inhibitor.
Further, methods are provided for limiting renal fibrosis in a patient. In one embodiment, the method involves reducing TGF-xcex21 activity in the patient while inhibiting xcex11xcex21 integrin receptors of the patent""s kidney cells. This activity can be reduced by administering to the patient an agent that irreversibly binds to TGF-xcex21 and inhibits its ability to bind with its receptor. Alternatively, this activity can be reduced by administering to the patient an agent capable of inhibiting the ability of TGF-xcex21 to transduce signals to the nucleus of a kidney cell.
In yet another embodiment, methods are provided for limiting matrix accumulation in the GBM of a patient with Alport Syndrome. In one embodiment, the method involves reducing TGF-xcex21 activity in the patient. This can be accomplished by administering TGF-xcex21 inhibitors as described herein.
In a particularly preferred embodiment, a method is provided for limiting renal fibroses by administering to a patient a calcineurin inhibitor, preferably tacrolimus.
Further, the present invention provides a mouse model for kidney disease wherein the mouse does not express a normal collagen type 4 composition in the GBM as a result of knocking out the collagen xcex13(IV) gene. That is, the mouse does not incorporate the collagen xcex13(IV), xcex14(IV), or xcex15(IV) chains into the glomerular basement membrane (thus the GBM is comprised entirely of collagen xcex11(IV) and xcex12(IV) chains, with respect to its type IV collagen chain composition). Furthermore, it does not express the xcex11xcex21 integrin receptor a result of knocking out the xcex11 subunit gene.
In the double knockout mouse there is delayed onset of proteinuria as compared to the prior art Alport mouse model. Furthermore, the animal lives nearly twice as long as Alport littermates. At about 8 weeks of age, which is the average age of death in Alport mice, the double knockout shows markedly reduced glomerular pathology. That is, as compared to Alport mice of the same age, the double knockout mouse has markedly reduced ultrastructural damage with far less GBM rarefication and very little effacement of the podocyte foot processes. Also, attenuated accumulation of fibronectin, laminin-1, and heparan sulfate proteoglycan in the GBM occur, while accumulation of entactin and type IV collagen are unchanged, relative to Alport mice. These results indicate that there is a specific role for the xcex11xcex21 integrin receptor in Alport renal disease pathogenesis. This is remarkable, considering that the single xcex11 integrin knockout has no obvious effect on renal physiology or function.
This mouse can be used for studying Alport syndrome, insulin dependent diabetes mellitus, and other disorders that are characterized by glomerulone phritis and/or fibrosis. This mouse can also be used for screening for agents that can be used to treat Alport syndrome and insulin dependent diabetes mellitus and other disorders that are characterized by deposition of extracellular matrix and/or fibrosis.